Skeletal tissue engineering is rapidly approaching the stage for human application despite the fact that rigorous preclinical testing to understand the cellular and environmental determinants of success or failure has not been developed. As traditional histological and molecular markers of tissue formation do not indicate the degree of bone formation, the knowledge of the proliferative properties of the progenitor cells that invest in the scaffold and the relative host/donor contributions to the repair has not been achieved. To overcome this problem, we have developed or acquired a series of GFP reporter transgenic mice that mark the source and level of differentiation of the major cellular components in bone repair. Murine models have been developed to assess these events utilizing 2D cryo-histology that preserves the activity of the reporters in mineralized tissues. However, details of temporal and spatial cell/scaffold interactions during the dynamic bone repair process are still not understood. Gaining this knowledge is vital for improving existing, or developing new research strategies and therapies. Thus, the primary objective of this application is to demonstrate to the tissue engineering community that it is possible to develop an in vivo 4D time-lapse imaging platform to visualize cell/scaffold/new bone interplay in a mouse calvarial defect using 2-photon microscopy. This will be the first time that a 3D cell- scaffold repair system is visualized in real-time with the emission of cell-specific GFP signals during the dynamic bone regeneration. We will then use this newly established platform to view different stages of bone development in two novel scaffolds created recently within our research group. The results will be used in an attempt to interpret prior observations, that lamellar scaffolds have superior bone forming ability to that of the cellular structure, irrespective of the type of progenitor cells seeded. We hypothesize that the way the osteoprogenitor cells initially distribute, proliferate and progress toward osteogenic differentiation in vivo determines the ultimate outcome of bone formation in a scaffold. Successful implementation of the 4D imaging platform will be transformative to the field because it will provide the essential information as to why a particular strategy succeeds or fails. The platform will become the primary imaging base of understanding the spatial relationships, for appreciating temporal events between the cellular elements the cellular elements and cell/scaffold interactions during bone formation. It will allow investigators to begin to understand and ultimately optimize scaffold design and cellular participants as well as growth factor selection and release profile design for early stages of skeletal regeneration. This knowledge base will be crucial for the bone tissue engineering community. PUBLIC HEALTH RELEVANCE: More than 1.3 million bone-repair procedures are conducted per year in the USA, which created a huge demand in bone re-generating materials. However, very little is known to date about the temporal and spatial interactions between scaffold and cells during the dynamic process of bone repair. In this study, we propose to establish a 4D in vivo imaging platform to visualize in real-time cellular activities and bone development in scaffolds, and thereby guide us to design better scaffold/cell complex for bone repair.